1. Field of the Invention
This invention relates to and has among its objects the provision of novel polysaccharide graft copolymer-enzyme products and methods for preparing them.
2. Description of the Art
Enzyme immobilization provides many important advantages over use of enzymes in soluble form, namely, enzyme reuseability, continuous operation, controlled product formation, and simplified and efficient processing. Continuous reactors utilizing immobilized enzymes provide high productivities and yields, and minimize downtime, enzyme costs, fermentor size, and capitol investment.
Conventional methods for immobilization of an enzyme on a solid substrate include covalent attachment of the enzyme to organic or inorganic carriers using cross linking agents such as glutaraldehyde; entrapment of the enzyme in a polymerizing polymer, and adsorption onto insoluble substrates such as ion exchange resins, activated charcoal, alumina, and the like. (For a detailed discussion, see Immobilized Enzymes by O. Zaborsky, CRC Press, 175 pages, 1973.)
Glucoamylase is one of the most important industrial enzymes. In the food and beverage industries, glucoamylase has been an important enzyme for starch saccharification because it can achieve complete breakdown of starch to glucose. In order to make the enzyme continuously reuseable, workers have immobilized it on various substrates but have encountered low enzyme activities, gradual deactivation of the enzyme, and low glucose yields (P. J. Reilly in Applied Biochemistry and Bioengineering 2: 185-207, Academic Press, Inc. (1979)). One of the reasons is the effect of slow diffusion of glucoamylase into the pores of the carrier substrate. Slow diffusion can affect not only the overall rate of hydrolysis but also the concentrations of intermediates as the reaction sequence proceeds (Lee et al., Biotechnology and Bioengineering 22: 1-17 (1980)). Low product yields can occur if high molecular weight starch fractions are excluded from small pores or if glucose, being slow to diffuse out of pores, forms reversion products such as maltose, isomaltose, and maltotriose. Further, temperature extremes arising from inadequate heat exchange are known to speed enzyme deactivation, especially in packed bed reactors.
H. Weetall in Immobilized Enzymes for Industrial Reactors, Ed., R. A. Messing, Academic Press, New York, pp. 207-212 (1975); Lee et al., 1980, supra, and Lee et al., Biotechnology and Bioengineering 18: 253-267 (1976) have indicated that many of these problems can be bypassed if reaction temperature is sufficiently low to maintain enzyme stability and if support diameter is sufficiently small to eliminate diffusion effects. Pilot scale operation, approximating these conditions, has shown promise for glucoamylase linked via glutaraldehyde to alkylamine porous silica (Lee et al., 1976, supra). Although glucoamylase activity was infinitely stable at the 38.degree. C. operating temperature, the reaction still showed evidence of diffusion limitation, and small yield losses of 1.5% glucose concentration were noted even though the catalyst particles were 300-600 .mu.m in diameter. While immobilization of glucoamylase by adsorption of the enzyme onto substrates such as ion exchange resins, DEAE-cellulose, CM-cellulose, CM-sephadex, and the like (Zaborsky, supra, pp. 80-81) is known, it is impossible to predict what substrates will bind and retain significant quantities of active glucoamylase and provide good product yield because adsorption of enzymes onto water-insoluble matrices depends on (1) the specific natures of both carrier substrate and enzyme and (2) is attributed to various mechanisms, e.g., ion exchange, physical adsorption, hydrophobic interactions (physicochemical bonding), van der Waals attractive forces, etc.